Bis-biguanides

ABSTRACT

The invention provides, as new compounds, salts of certain bactericidally active bis-biguanides bases with sequestering amino-carboxylic acids, the compounds being useful as bactericides. The invention also provides compositions containing these salts; the compositions preferably incorporate quaternary ammonium compounds as solubilizing agents for the salts.

United States Patent [191 Stephenson et al.

[45] June 10, 1975 BIS-BIGUANIDES [75] Inventors: Ronald Arthur Stephenson,

Henley-on-Thames, England; Bente Lissy Laursen, Ektorp; Ove Henning Mattsson, Taeby. both of Sweden [73] Assignee: Kemanord Aktiebolag, Sweden [22 Filed: May 18, 1972 [2]] Appl. No.: 254,440

[30] Foreign Application Priority Data May 18. 1971 Sweden 6431/71 [52] US. CL... 260/501.ll; 26()/501.14; 260/290 R;

424/3161424/326; 260/501.l5 [51] Int. Cl. C07c 101/00 [58] Field of Search zoo/501.11, 501.14

[56] References Cited UNITED STATES PATENTS 3.271863 9/1966 Cutler et a1 26()/5()l.l4 X

3,468,898 9/1969 Cutler et al 260/501.l4 X

FOREIGN PATENTS OR APPLICATIONS 2,158,102 5/1972 Germany OTHER PUBLICATIONS Brown et al., Nature, Vol. 207 (No. 5004), p. 1391-1393 (1965).

Primary Examiner-Leon Zitver Assistant ExaminerGeorge T. Breitenstein Attorney, Agent, or Firm-Cushman, Darby & Cushman [57] ABSTRACT 1 Claim, 4 Drawing Figures PATENTEDJUH 10 I975 SHEET com com

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PATENTEDJUH 10 I975 SHEET com 256 a 5535 No BIS-BIGUANIDES This invention concerns novel bis-biguanides, processes for their preparation, and compositions incorporating them. More specifically, the invention relates to certain novel bis-biguanide salts which appear to have interesting bactericidal properties, and which may thus be of use in human or veterinary medicine.

Bis-biguanides are known (in general) to have certain bactericidal properties, and one such compound in particular l,6-bis-(N -p-chlorophenyldiguanido-N hexane, which has the open name chlorohexidine has found considerable use in medicine as a bactericide, and primarily as a general disinfectant. Unfortunately, however, the known bis-biguanide bases and most of their salts are only sparingly soluble in water (which is the solvent of choice for making up disinfectant compositions), and therefore have a limited use from a commercial point of view.

We have now found that a new class of bis-biguanide salts can be dissolved in water more easily than most known salts, and is more active than all known salts and thus these compounds are more useful as disinfectants in particular and bactericides in general.

In one aspect, therefore, this invention provides a salt of a known bactericidally active bis-biguanide base with a sequestering amino-carboxylic acid falling within the general formula:

wherein:

R represents hydrogen, an alkyl group, a carboxymethyl group, a hydroxyethyl group or a hydroxypropyl group;

R represents a carboxymethyl group, a hydroxy ethyl group or a hydroxypropyl group; and

either Z represents the group [CH ,.CH .N(R)],,- (wherein R is defined as R, though is not necessarily identical thereto, and n is 0,1,2 or 3), or Z represents the group (Ill) (wherein R" is as defined hereinbefore), subject to the proviso that, when n is l R,, R and R are carboxymethyl groups.

Typical amino-carboxylic acids falling within general formula II are:

N,N'-ethylenediaminediacetic acid (EDDA) n l, R and R hydrogen, and R carboxymethyl;

N-hydroxyethylethylenediaminetriacetic acid (HEDTA) n l, R and R carboxymethyl, and R Z-hydroxycthyl;

N,N-dihydroxyethylenediaminediacetic acid (HEDDA) n l, R and R Z-hydroxyethyl, and R carboxymethyl;

diethylenetriaminepentaacetic acid (DTPA) n 2, and R, R and both R"s carboxymethyl;

l,2-diaminocyclohexanetetraacetic acid (CDTA) R, R and R carboxymethyl;

nitrilotriacetic acid (NTA) n 0, and R and R carboxymethyl;

N-hydroxyethyliminodiacetic acid (HIMDA) n 0, R 2-hydroxyethyl, and R carboxymethyl;

N,N-dihydroxyethylaminoacetic acid (DAA) n O and R and R 2-hydroxyethyl;

iminodiacetic acid (IMDA) n O, R hydrogen and R carboxymethyl; and

3-hydroxypropyliminodiacetic acid (HPIMDA) n 0, R 3-hydroxypropyl, and R carboxymethyl.

Of these particular aminocarboxylic acids, HEDTA DTPA, NTA and DAA are preferred, and HEDTA and DAA are especially preferred.

The salts of this invention the compounds may essentially be regarded as the reaction product between the acidic sequestering amino-carboxylic acid and the basic bis-biguanide (and can, thus, be referred to generally not only as salts but also as sequestrates) may contain the two compounds (the acid and the base) in varying proportions, because the acids are mostly poly-acidic (they have two or more acid groups) and chlorhexidine is dibasic, and because all of the basic/acidic groupings may not necessarily be used up. However, in general (but not always) the acidic amino-carboxylic acids tend to act either as monoacidic acids or as di-acidic acids (wherein either one or two acidic groupings are used).

Thus, for example, the particularly preferred aminocarboxylic acid HEDTA acts either in a monoor diacidic fashion, and so far as can be determined the only salts formed are chlorhexidine mono- HEDTAcetate (wherein the HEDTA is di-acidic) and chlorhexidine di-HEDTAcetate (wherein the HEDTA is mono-acidic).

Of the various salts (sequestrates) obtainable usin the preferred amino-carboxylic acids listed above, the particularly preferred ones are:

mono-chlorhexidine nitrilotriacetate /NTA); -tri-chlorhexidine di-[diethylenetriaminepentaacetate] (3 chlorhex/2 DTPA);

mono-chlorhexidine di-[N,N-dihydroxyethylaminoacetate] (chlorhex/2 DAA); and, especially, mono-chlorhexidine N-hydroxyethylethylenediaminetriacetate (chlorhex/HEDTA), and monochlorhexidine di-[N-hydroxyethylethylenediaminetriacetate] (chlorhex/2 HEDTA).

The sequestrates of this invention may be made by any of the conventional methods employed for the preparation of salts. In particular, they may be prepared by a straight acid/base reaction, or they may be prepared by a metathesis (or double decomposition) reaction between another salt of the acid and another salt of the base.

Accordingly, in another aspect this invention provides a process for the preparation of a bisbiguanide/amino-carboxylic acid sequestrate of the invention, in which chlorhexidine (or salt thereof) is reacted in an aqueous medium with an appropriate amino-carboxylic acid (or salt thereof) to give the desired sequestrate.

While the amino-carboxylic acids (and their salts) are in general water-soluble, the bis-biguanide (and its salts) tend to be not very soluble in water. Accordingly it is preferred that the reaction be effected in the pres- (chlorhexence of a medium in which the bis-biguanide is soluble, which medium is easily miscible with the water present. Such mediums are the alkanols, and particularly suitable alkanols are methanol and ethanol. Thus, the reaction is preferably effected in aqueous methanol or ethanol.

Of course, both the bis-biguanide and the aminocarboxylic acid may be dissolved in the aqueous alka' no], or the former may be dissolved in alkanol, the latter in water, and the two solutions mixed.

When effecting the reaction using a salt of the his biguanide and a salt of the amino-carboxylic acid (metathesis) the former is preferably one of these with common organic acids (for example, the diacetate) and the latter is preferably an alkali-metal salt (for example, a sodium salt). Mineral acid bis-biguanide salts are to be avoided since they are extremely water-insoluble.

The formed salts may be isolated by conventional techniques, but for some purposes they need not be isolated at all (the prepared solution containing them may be used as it is), and this is discussed hereinafter.

As stated above, the novel bis-biguanide sequestrates of this invention appear to exert a useful anti-bacterial activity. Now, while the bis-biguanides of general formula l are known to have anti-bacterial activity in salt form, the sequestrates derived from them have a greater anti-bacterial activity and this can be manifest in several ways. Firstly, the spectrum of activity of the bis-biguanide salts at a given concentration can be extended"; that is to say, a given bis-biguanide salt may be inactive against a particular species of bacteria at a given concentration, whereas a corresponding sequestrate will be active when applied at the same concentration expressed in terms of the bis-biguanide base. Secondly, if a particular concentration of a bis-biguanide salt is needed to kill a bacterial culture, then a lower concentration of a corresponding sequestrate may be needed to have the same killing power. Thirdly, since many of the known salts of the bis-biguanides are only moderately soluble in water, it may be possible 'to reach a greater concentration in terms of bis-biguanide base using the more soluble sequestrates.

However, before any of the sequestrates of this invention may be used in medicine, they should preferably be formed into compositions by association with suitable vehicles. Naturally, the term suitable vehicle isused herein to exclude any possibility that the nature of the vehicles, considered of course, in relation to the use to which the composition is to be put, could be harmful rather than beneficial. The choice of a suitable mode of presentation, together with an appropriate veprobably by far the most important use of the compositions will be as disinfectants, primarily for use in hospitals, and as such the composition will generally be liquid, the vehicle being, for instance, water. However, the sequestrates may well find other uses (not only in medicine but also in any field where a bactericide is requested such as in the food trade), and indeed in some respects they seem adapted not only for use as disinfectants of inanimate objects (bed linen, walls, tables, pots, pans and so forth equipment in general) but also antiseptics on or in humans and other animals. Thus, in general the compositions of this invention may be administered orally, perlingually, topically, or rectally, and in respect of these modes, the vehicle is preferably:-

a. the ingestible excipient of a tablet, coated tablet, sublingual tablet or pill; the ingestible container of a capsule or cachet; the ingestible pulverulent solid carrier of a powder; or the ingestible liquid medium of a syrup, solution, suspension or elixir;

b. the solid or liquid carrier medium of an ointment, paste, gel, salve, lotion, balm, unguent, wash, cream, solution, emulsion or dusting powder;

c. a base material of low melting point capable of releasing an active ingredient to perform its pharmacological function, which base material when appropriately shaped forms a suppository.

Whilst the modes of presentation just listed represent those most likely to be employed, they do not necessarily exhaust the possibilities.

The compositions of this invention that are prepared by formulating an isolated sequestrate will, naturally, contain the two moieties involved the bis-biguanide moiety on the one hand, and the amino-carboxylic acid moiety on the other hand in stoichiometric molar proportions. Thus, for example, a composition made from chlorhex/HEDTA contains the two moieties in the molar ratio 1:1, while a composition made from chlorhex/2 HEDTA contains the two moieties in the molar ratio 1:2.

It is possible, however, to make up compositions containing an excess of either moiety (or, rather, containing an additional quantity of either the bisbiguanide itself or the amino-carboxylic acid itself), and in many cases the use of an excess particularly an excess of amino-carboxylic acid is advantageous and appears to give rise to somewhat better bactericidal properties than might be expected. With the aminocarboxylic acid, for example, excesses of the order of half a mole per mole of bis-biguanide give compositions which perform quite satisfactorily.

The sequestrates of this invention, though in general all highly active as bactericides, are not all equally soluble in water. Indeed, some of them are so poorly soluble that it is difficult, if possible at all, to form a liquid aqueous composition that contains enough sequestrate to act properly as a bactericide without using a solubilizing agent (discussed hereinafter). Accordingly, this invention does not extend to sequestrate-containing compositions which are aqueous and liquid, and which do not incorporate a solubilizer, where the sequestrate is incapable, without the aid of a solubilizer, of being dissolved in water to a concentration of more than 1000 ppm.

In order to prepare a commercially attractive liquid aqueous composition according to the invention (as opposed to a composition which works, as a bactericide, though rather poorly) it is necessary to be able to prepare and sell a concentrate, and thus to dissolve large quantities of the sequestrate (and by large quan tities are meant amounts of from l percent by weight of the composition upwards). In order, therefore, to ensure that the compositions of the invention meet this criterion it is highly desirable that they should contain, in addition, a solubilizing agent for the sequestrate, and a class of solubilizing agents which is particularly suitable (partly because most of the agents are themselves bactericidal, partly because the agents have detergent properties useful in a disinfectant composition and partly because, as is discussed below, the thus-formed compositions appear to exhibit synergism) is the quaternary ammonium compounds.

Of the very large number of available quaternary ammonium compounds (QACs) the following seem preferable:

those having a cation of the general formula CH3 (lV) (wherein:

Y is an alkyl group containing from 8 to carbon atoms, a di-isobutyl-phenoxy-ethoxyethyl group, or a diisobutyl-cresoxyethoxyethyl group; and

Y is an aralkyl group, preferably a benzyl group, optionally substituted in the aromatic nucleus) especially those having the benzalkonium cation (which is described in the literature as alkyldimethylbenzyl ammonium wherein the alkyl is the well known cocogroup being a mixture of alkyl groups mainly in the C -C range such as lauryl, myristyl, etc.), and the compounds sold under the trade names Hyamine LOX and Hyamine 1622 in the USA (which are described as having the cations di-isobutylcresoxyethoxyethyl dimethyl benzyl ammonium and di-isobutylphenoxyethoxyethyl dimethyl benzyl ammonium), and the myristyldimethylbenzyl ammonium compounds;

those having a cation of the general formula CH Yj-CH;,

(wherein:

(wherein:

Y and Y which may be the same or different, are each an alkyl group containing from 8 to 18 carbon atoms), especially the didecyldimethyl ammonium and the dioctyl-dimethyl ammonium cations,

those having a cation of the general formula monium, .cetyltrimethyl wherein:

Y isan alkyl group of 8 to 20 carbon atoms; and Y and Y are the same or different, and each is a hydroxy-substituted alkyl or hydroxyalkoxyalkyl group), especially alkyl dihydroxyethyl) benzyl ammonium, wherein the alkyl is, for example, a cocogroup; andthose having a cation of the general formula 9 Y (vm) (wherein: Y is an alkyl group of 8 to 20 carbon atoms), especially the cetylpyridinium cation.

The particularly preferred quaternary ammonium cations are benzalkonium, myristyldimethylbenzyl amammonium, and coco dihydroxyethyl)benzyl ammonium.

Incidentally, it does not seem to matter very much what anion is associated with the quaternary ammorules about how much quaternary is required to solubilize how much sequestrate. Nevertheless, by way of general indication it may be said that a good solubilizing quaternary for example, Cetrimide (cetyl trimethyl ammonium bromide) can solubilize as much sequestrate as will give a 20 percent by weight aqueous solution, and at that level is solubilizing on an equal weight basis (whereas at lower concentrations of sequestrate the quaternary tends to solubilize slightly more than its own weight). A not-so-good solubilizing quaternary may only solubilize as much sequestrate as will give a 5 percent solution, and at that level may be solubilizing perhaps only l/ 10 its weight of sequestrate. A few general examples of maximum solubilizing power of various quaternary ammonium compounds with various bis-biguanide sequestrates are as follows (the percentages are by weight based on the final solution, and the formed solutions were stable):

18 percent centrimide solubilizes 27 percent chlorhex/HEDTA: 30 percent benzalkonium chloride solubilizes 15 percent chlorhex/HEDTA; 20 percent benzalkonium bromide solubilizes 20 percent chlorhex/HEDTA; 20 percent centrimide solubilizes 30 percent chlorhex/NTA; 20 percent centrimide solubilizes 30 percent 3 chlorhex/2 DTPA.

In any given case the solubilizing power of a particular quaternary to a particular sequestrate may easily be determined by trial.

As stated above, one of the reasons for choosing quaternary ammonium compounds as the solubilizing agents is that not only are the quaternaries usually bactericidal in their own right but also there appears to be synergistic action between the quaternary and the sequestrate, the bactericidal activity of the composition being greater (in some cases considerably greater) than might be expected from a simple summation of the activities of the two components. Of course, it is in marginal cases difficult to be sure that synergy occurs but, as is pointed out hereinafter in connection with the Test Results, in some cases there would seem to be no such difficulty.

The compositions of this invention may be made simply by mixing the sequestrate with the vehicle (and with excess sequestrate moiety and/or with quaternary ammonium compound as appropriate). However, as intimated above the formed sequestrates need not necessarily be isolated from their reaction media; the reaction solution may well constitute a suitable aqueous liquid composition.

It follows, therefore, that compositions of this invention can be prepared simply by dissolving the bisbiguanide and the amino-carboxylic acid in water or an aqeous alkanol to which also may be added (ifdesired) the quaternary ammonium compound.

Furthermore, because of the ability of the quaternary ammonium compounds to form salts with the aminocarboxylic acids (the salts being quaternary ammonium sequestrates), it is also possible to form compositions of this invention by dissolving in water or an aqueous alkanol the bis-biguanide and the quaternary ammonium sequestrate.

Examples of these various procedures are given hereinafter.

The following Examples are now given, though by way of illustration only, to show details of preferred compounds, preparative processes and compositions according to this invention.

PART I: PREPARATION OF BlS-BIGUANIDE SEQUESTRATES EXAMPLE 1: Mono(chlorhexidine) mono(hydroxyethylethylenediamine triacetate Method A Method B 25 ml of a 0.01 M solution of chlorhexidine in methanol (thus, 0.1263 grammes of chlorhexidine) were added to 0.0695 gm of HEDTA in 35 ml of distilled water. Thus, the acid and base were present in equimolar proportions. This solution was evaporated to half volume on a steam bath, then allowed to cool slowly and, after standing for about 2 hours, crystallization was initiated by scratching the side of the vessel with a glass rod. The precipitate was filtered and dried, to give the required salt.

Method C l Gm of chlorhexidine diacetate was dissolved in 25 mls of industrial methylated spirit, and was added to a solution of 0.4448 grams of HEDTA in 3.2 mls of normal sodium hydroxide and 5 mls of distilled water. Thus, the acid and base were present in equimolar proportions. A precipitate formed on standing which was filtered, washed with distilled water and dried to give the desired compound.

In each case, a differential scanning calorimeter showed that salt formation had occurred. This was confirmed by the melting points, which were as follows:

Melting Points HEDTA 156C Chlorhexidine 127C Salt (Methods A and B) 173C (Method C) 177C Physical mixture of acid and 124C base EXAMPLE 2: Mono(chlorhexidine) di( hydroxyethylethylenediaminetriacetate) 1.33 G of chlorhexidine and 1.42 g HEDTA were dissolved together in 25 ml distilled water by heating. Thus, the molar ratio of base to acid was 1:2. The solution was cooled, whereupon a white precipitate formed, which was filtered, washed and dried to give mono(chlorhexidine) di(hydroxyethylethylenediaminetriacetate). The pH of the mother liquor was 6.1, the melting point of the salt was 172C and its solubility in water was 0.3 percent w/w at 20C.

EXAMPLE 3: Mono(chlorhexidine) mono(nitrilotriacetate) 25 ml of a 0.04 M solution of chlorhexidine diacetate in distilled water were added to 25 ml of a 0.04 M solution of disodium nitrilotriacetate in distilled water. Thus, the acid and base were present in equimolar proportions. A fine, oily precipitate was formed which at first remained suspended in the liquid, but which solidified on standing. This precipitate was filtered, washed with distilled water and dried to give mono(chlorhexidine) mono(nitrilotriacetate). The solubility of the salt in water at 20C is 0.3% w/w,

A differential scanning calorimeter confirmed the formation of the salt and this was further verified by melting point tests, which gave the following melting points:

NTA 225C Chlorhexidine 127C Salt 176C Physical Mixture of NTA and chlorhexidine 129C EXAMPLE 4: Tri(chlorhexidine) di(diethylenetriaminepentaacetate) 0.69 G of diethylenetriaminepentaacetic acid was dissolved in ml of distilled water by heating. 1.33 g of chlorhexidine was dissolved in this solution by heating and stirring. Thus, the base and acid were present in a molar ratio of 322. After cooling an oil separated, but after two days at room temperature all the oil had turned into white crystals. The crystals were recrystallized in water, dried at 100C to constant weight, to give tri(chlorhexidine) di(diethylenetriaminepentaacetate). This compound had a melting point of 157C and a solubility in water of 0.2 percent w/w at 20C.

EXAMPLE Mono(chlorhexidine) di( dihydroxyethylaminoacetate) 100 ml of a 2 percent solution of chlorhexidine diacetate in distilled water were added to a solution of 1.234 g of the monosodium salt of dihydroxyethylaminoacetic acid (equivalent to 1.0432 grammes of dihydroxyethylaminoacetic acid) in distilled water. Thus the chlorhexidine cation and the dihydroxyethylaminoacetic acid) anion were present in a molar ratio of 1:2. The precipitate was filtered, washed and dried, to give mono(chlorhexidine) di( dihydroxyethylaminoacetate). The solubility of this salt in water 0.2 percent w/w at 20C.

A differential scanning calorimeter showed the formation of the salt, and this was confirmed by melting point tests, which gave the following melting points:

Dihydroxyethylaminoacetic acid 180C Chlorhexidine 127C Salt 86C Physical Mixture 107C Part II: Compositions without quaternary ammonium compounds EXAMPLE 6. Mono( chlorhexidine) di(hydroxyethylethylenediaminetriacetate a. Preparation of the compositions Solid mono(chlorhexidine) di(hydroxyethylethylenediaminetriacetate), prepared as in Example 2, was dissolved in standard hard water (W.H.O. formulation see below) to yield solutions containing 400, 600, 800, 1200, 1600, 2000 and 2500 ppm of the salt.

b. Bactericidal Tests The solutions prepared in a) were tested for bactericidal activity according to British Standard No. 3286: 1960 (see the publication Method for laboratory evaluation of disinfectant activity of quaternary ammonium compounds by suspension test procedure, published by the British Standard Institution, British Standards House, 2 Park Street, London, S.W.1. London, 1960).

Many anti-bacterial compositions are less active against gram negative bacteria than against gram positive bacteria. Furthermore, their anti-bacterial activity is reduced in the presence of hard water and/or organic matter such as blood serum.

Thus a particularly stringent anti-bacterial test is one against the highly resistant gram negative bacterium, Pseudomonas pyocyanea in the presence of hard water.

The essential details of the test procedure are as follows:

i. Materials Nutrient Broth Nutrient Broth No. 2 is used which contains:

Lab-lemco (A proprietory brand of proteinaceous grams material) Peptone 10 grams Sodium Chloride 5 grams Distilled water to 1,000 mls.

Standard Hard Water (W.H.O. Formulation) 10% CaCl 6H O (wt/vol) 10% Mg SOJH O (wt/vol) Distilled water 17.5 mls. 5.0 mls. 3,300 mls.

This is autoclaved at lbs. per square inch pressure for 15 minutes. Disinfection Time 5 Minutes. Temperature 22C. Test organism Pseudomas aeruginosa (Pseudomonas pyocyanea). N.C.T.C. No. 6749 Organic matter added 10 percent bovine serum. ii. Preparation of Culture A culture from agar of the organism was prepared in nutrient broth and incubated at 37C for 24 hours. Daily subcultures were continued, and cultures between the third and seventh generation used for the test. The broth culture of Pseudomonas pyocyanea was filtered aseptically before use, or, shaken, allowed to settle for 15 minutes and the supernatant liquor used for the test. Culture dilution One part of the broth culture was diluted with 24 parts of Standard Hard Water, that is 4 ml. broth culture was added to 96 ml. of Standard Hard Water. The average-count of bacteria in such a dilution is 5 X 10 organisms per ml. Method of Test Serial double strength dilutions of the anti-bacterial compositions to be tested were made in standard hard water and 2.5 ml. transferred to a sterile test tube. (By serial double strength dilutions we mean that to test a compound at, for example, 500 ppm, one must prepare a strength 1000 ppm since in the following step the volume in the tube is doubled). Starting at zero time 2.5 m1 of the diluted bacterial culture suspensions were added to each test tube containing the anti-bacterial composition under test, (the contents of the bacterial culture/anti bacterial composition mixture were adjusted to contain 300 ppm hardness). After 5 minutes 1 ml of the bacterial culture mixture from each tube was transferred to a sterile tube containing 9 ml of inactivator. 1 M1 of the mixture thus obtained was transferred to an agar plate, and a further 1 ml to a second tube containing 9 ml of inactivator. Similarly 1 ml of the mixture produced in the second tube was transferred to an agar plate and 1 ml to a third tube containing 9 ml of inactivator and so on. The inactivator stopped the further action of the anti-bacterial agent, and each live bacterium left on addition to the activator produces a colonly on the agar plate. The number of colonies on each plate was counted, multiplied by the Table I Final concentration of bactericide p.p.m. 200 300 400 600 Bactericide Mono(chlorhexidine) Di(I-IEDTA) 21,818 2,909 16 Chlorhexidine dihydrochloride (control) u/c u/c Chlorhexidine digluconate (control) u/c u/c u/c u/c u/c number of survivors too great to be counted.

These results show that the quantity of mono(chlorhexidine).di(I-IEDTA) required to kill a given quantity of Pseudomonas pyocyanea is markedly less than that of either chlorhexidine dihydrochloride or of chlorhexidine digluconate.

EXAMPLE 7: Mono( chlorhexidine) mono(hydroxyethylethylenediaminetriacetate a. Preparation of the compositions Solid mono(chlorhexidine) mono(hydroxyethylethylenediaminetriacetate, prepared as in Example 1, was dissolved in standard hard water (W.H.O. formulation) to yield solutions containing 400, 800, 1200, 1600 and 2000 ppm of the salt.

b. Bactericidal tests The solutions prepared in a) were tested for bactericidal activity in the same manner as in Example 6. Control examples were provided using the same concentrations of chlorhexidine digluconate.

Results The results are shown in Table II below.

TABLE II Final concentration of bactericide p.p.m. 200 400 600 800 1000 Bactericide Mono (chlorhexidine) Mono (HEDTA) 390 0 0 0 0 Chlorhexidine Digluconate (control) 300 540 26 These results show that the bactericidal properties of mono(chlorhexidine) mono(I-IEDTA) are markedly superior to those of chlorhexidine digluconate.

EXAMPLE 8: Mono( chlo rhexidine) mono( nitrilo-triacetate a. Preparation of the compositions Solid mono(chlorhexidine) mono(nitrilo-triacetate), prepared as in Example 3, was dissolved in standard hard water (W.H.O. formulation) to yield solutions containing 400, 800, 1200, 1600 and 2000 p.p.m. of the salt.

b. Bactericidal Tests The solutions prepared in a) were tested for bactericidal activity in the'same manner as in Example 6. Control examples were provided using the same concentrations of chlorhexidine digluconate.

Results insoluble The results are shown in Table III below.

TABLE III Final concentration of bactericide p.p.m. 200 400 600 800 1000 Bactericide Mono(chlorhexidine) Mono( nitrilotriacetate) Chlorhexidine Digluconate (control) 300 540 26 These results show the superiority of mono(chlorhexidine) mono(nitrilotriacetate) to chlorhexidine digluconate as a bactericide.

EXAMPLE 9: Tri(chlorhexidine) di(diethylenetriaminepentaacetate) a. Preparation of the compositions Solid tri(chlorhexidine) di(diethylenetriaminepentaacetate) prepared as in Example 4 was dissolved in standard hard water (W.H.O. formulation) to yield solutions containing 400, 800, 1200, 1600 and 2000 p.p.m. of the salt. b. Bactericidal tests The solutions prepared in a) were tested for bactericidal activity in the same manner as in Example 6. Control examples were provided using the same concentrations of chlorhexidine digluconate. Results The results are shown in Table IV below.

TABLE IV Final concentration Q of bactericide p.p.m. 200 400 600 800 I000 Bactericide TABLE lV- Continued Final concentration of bactericide p.p.m. 200 400 600 800 1000 Tri(chlorohexidine) DI(DTPA) u/c 26.36 B3 0 chlorhexidine Digluoconate (control) 4624 u/c 3l8 These results show that tri(chlorhe xidine) di(diethylenetriaminepentaacetate) is markedly superior to chlorhexidine digluconate as a bactericide.

EXAMPLE Mono(chlorhexidine) di(hydroxyethylaminoacetate).

TABLE V Final concentration of bactericide 400 600 800 1000 di(hydroxyethylaminoacetate) p.p.m.

Bactericide Mono(chlorhexidine) Di(hydroxyethylaminoacetate) 600 35 0 0 Chlorhexidine Digluconate (control) 1200 520 308 These results show that mono(chlorhexidine) di( hydroxyethylaminoacetic acid) is markedly superior to chlorhexidine digluconate as a bactericide.

PART III: COMPOSITIONS CONTAINING QUATERNARY AMMONIUM COMPOUNDS EXAMPLE l1: Mono(chlorhexidine) mono(l-IEDTA) lCetrimide a. Preparation of the composition 14.3 G of chlorhexidine, 20.4 g of cetyltrimethyl ammonium bromide and 7.8 g of hydroxyethylethylenediaminetriacet'ic acid (HEDTA) were allowed to react in 57.5 g water'under agitation, until a clear solution was obtained which was dilutable with water in all concentrations and contained a mixture of mono(- chlorhexidine) mono(hydroxyethylethylenediaminetriacetate) and cetyltrimethylammonium bromide.

b. Bactericidal tests (Antiseptic Test) The solution obtained in a) was tested for bactericidal activity according to British Standard Specification 2462.

As stated above, a particularly stringent antibacterial test is against the highly resistant gram negative bacterium Pseudomonas pyocyanea in the presence of hard water and organic matter. The essential details of the test are set out below:

i. Materials Nutrient Broth Nutrient Broth No. 2 is used as in Example 6 above. Inactivator Nutrient Broth with the addition of 0.5 percent of lecithin (ex ovo) a substance derived from egg yolks, being a compound of choline, glycerol, phosphoric acid and various fatty acids) and 2 percent Lubrol W. a proprietory brand of an anhydrous condensation product of a long chain fatty alcohol and ethylene oxide). The pH is adjusted if necessary to 7.2. Bacteria i Pseudomonas aeruginosa (pyocyanea) N.C.T.C. No. 6749 maintained on nutrient agar.

ii. Preparation of Culture A culture from agar of the organism was prepared in nutrient broth, and incubated at 37C for 24 hours. Daily subcultures were continued, and cultures between the third and seventh generation were used for the test. The broth culture of Pseudomonas pyocyanea was filtered aseptically before use, or shaken, allowed to settle for 15 minutes, and the supernatant liquor used for the test. Culture dilution One part of the broth culture was diluted with 24 parts of distilled water, i.e. 4 ml. broth culture 96 ml of distilled water. The average count of bacteria in such a dilution would be 5 X 10 organisms per ml. Method of Test Serial double strength dilutions of the anti-bacterial compositions to be tested are made in distilled water, and 2.5 ml transferred into sterile test tubes. Starting at zero time 2.5 ml of the diluted bacterial culture suspensions containing 20 percent of bovine serum were added to each test tube containing an anti-bacterial composition under test After 5 minutes a loop full (about 0.1 ml) of the bacterial culture/anti-bacterial composition mixture from each tube was transferred to 10 ml of the inactivator broth medium in a new series of sterile test tubes. After a total of 10 minutes another loop was transferred from the original bacterial culture/anti-bacterial composition mixture to a similar new set of tubes containing inactivator broth medium. The inactivator stopped the further action of the antibacterial and allowed the bacteria to grow again.

All the tubes containing the inactivator broth seeded with bacteria transferred on the loop were incubated at 37C for 48 hours, and examined for bacterial growth. The result was recorded as positive where growth occured and negative where no growth occurred. The lowest concentration of antibacterial which gave a negative result after the 5 minute contact test can be seen 15 16 Dilution Transfer in minutes dine, 18.2 g of cetyltrimethylammonium bromide and 5 l 1 1.2 g of hydroxyethylethylenediaminetriacetic acid in 53 g water. l-666 1,500 m 14000 1,000 SB... b. Bactericidal Tests 3383 f 238 ppm 5 The composition prepared in a) was tested for bacppm terlcidal effects in the same manner as in Example 11. v The results are shown in Table VII below.

Concentration giving 100 percent kill 1,500 ppm. TABLE VII It will be appreciated that the true figure may be anywhere between ],QOO ppm and 1,500 ppm, and thus the Killing Dilution killing concentrations given in the followmg tests are subject to errors of substantially the same magnitude. Baclericide gPP )PP q lpp PP Organic One can determine the concentration of the various g'g e ffiggf gg z m gfifg mixtures made according to the Examples (expressed in terms of parts per million of total organic material z g g g [bis-biguanide plus quaternary ammonium compound p p 75 75 45 195 aminocarboxylic acid]) and hence, knowing the relalive quantities of Components, One-Can Calculale These results should be compared to those given for from the Antiseptic Test results the killing dilution in hl h i i digluconate and Q AC sequestrate in terms of bls'biguamde and/0T quaternary ammonium Table VI above, whereupon it will be seen that the compoundcomposition of this invention is markedly superior as a Results I bactericide to both these conventional agents.

The result are shown in Table VI below. To provide controls, the killing dilutions of tests on chlorhexidine EXAMPLE 14! digluconate and a typical quaternary ammonium sea preparation f the composition questrate mono(myrrstyldimethylbenzylammomum) 15 g of the di-(myristyldimethylbenzyl ammonium) m0I'l0(HEDTA) were also tested salt of HEDTA and 8 g of chlorhexidine were dissolved TABLE VI Killing Dilutions Bacteri- (a)ppm bis- (b)ppm quar- (c)ppm acid ppm organic cide biguanide ternary ammomaterial base nium compound (a)+(b)+(c) (cation anion) Composition of this invention 60 90 180 Chlorhexidine Digluconate (control) 200 150 350 QAC sequestrate (control) 1060 440 1500 These results show the superiority of the composition in 77 g water, and an aqueous Solution was obtained.

of the present invention to chlorhexidine digluconate and the QAC sequestrate as a bactericide.

EXAMPLE l2: Mono(chlorhexidine) mono(HEDTA) lCetrimide b. Bactericidal tests The composition obtained in a) was tested for bactericidal activity in the same manner as in Example 11, and the results are shown in Table VIII below.

A solution of 12.6 g of chlorhexidine, 18.8 g of cetyl- TABLE VI trimethylammonium bromide and 9.2 g of hydroxyethylethylenediaminetriacetic acid in 300 g water was Killing Dilution heated to C and mixed with 8 parts of glycerol under agitation. A completely gelatinized mixture was 60 Ba cteritggppmlgis- (b)ppm quar- (c)ppim ppm or arliic obtained which could be used for application to the e gg e $3 2 2 32 aci (am-113i 0) skin. (cation anion) EXAMPLE l3: Mono(chlorhexidine) mono(l-IEDTA) ompolCetrimlde smon a. Preparation of the composition {ga 53 7] 29 153 A solution was prepared from 17.6 g of chlorhexi- These results should be compared to those given for chlorhexidine digluconate and QAC sequestrate in Table VIII above, whereupon it will be seen that the composition of this invention is markedly superior as a bactericide to both these conventional agents.

EXAMPLE 15: Mono(chlorhexidine) di(I-IEDTA) di(myristyldimethylbenzylammonium) mono(I-IEDTA) Myristyldimethylbenzylammonium hydroxide was prepared using essentially the following method. 367.5 Gm (1 mole) of myristyldimethylbenzylammonium chloride was dissolved in the smallest possible quantity of isopropyl alcohol. A saturated solution of 56 gm (1 mole) of potassium hydroxide in absolute ethanol was added under agitation. After a few hours, the precipitate of potassium chloride which had formed was filtered off and washed with isopropyl alcohol, the washings being returned to the main solution. The resultant alcoholic solution contained myristyldimethylbenzylammonium hydroxide. This solution was divided into aliquots each containing 0.1 mole (34.9 gm) of the quaternary ammonium hydroxide. The mono(myristyldimethylbenzylammonium) salt of hydroxyethylethylenediaminetriacetic acid was prepared by taking one of the aliquots containing 0.1 mole of myristyldimethylbenzylammonium hydroxide and adding 27.8 gm (0.1 mole) of a solution of HEDTA in water, then making the solution up to 418 ml. This percent w/w solution of the quaternary ammonium sequestrate contained 8.18 percent of the quaternary ammonium cat- EXAMPLE l6: Mono(chlorhexidine) mono(I-IEDTA)(Myristyldimethylbenzylammonium hydroxide) a. Preparation of the composition By the same method as in Example 15, di(myristyldimethylbenzylammonium) mono(hydroxyethylethylenediaminetriacetate) was prepared by using 0.1 mole of the quaternary ammonium hydroxide and 13.9 gm (0.05 mole) of HEDTA, and this was made up to 314 ml to give a 15 percent w/w solution containing 10.6 percent of quaternary ammonium cation and 4.4 percent of dibasic HEDTA anion.

By taking 2 ml of this 15 percent di(myristyldimethylbenzylammonium) mono(hydroxyethylethylenediaminetriacetate) and 0.16 gm of chlorhexidine base a 1:1 compound of chlorhexidine to dibasic HEDTA anion was prepared, that is mono(chlorhexidine) mono(hydroxyethylethylenediaminetriacetate). The result was a clear solution which could be further diluted to a clear solution containing 1060 ppm quaternary ammonium cation, 440 ppm HEDTA anion and 800 ppm of chlorhexidine.

b. Bactericidal tests The composition prepared in a) above was tested for bactericidal activity in the same manner as in Example 11 above. To provide controls, the killing dilutions of chlorhexidine digluconate and di(myristyldimethylbenzylammonium) mono(hydroxyethylethylenediaminetriacetate) were also tested. The results are shown in Table 1X below.

TABLE IX Killing Dilutions Bactericide ppm ppm ppm ppm ppm of Chlorhexidine Total Quaternary Quaternary HEDTA chlorhexidine active by weight of organic Ammonium Ammonium anion ingredients active matter compound cation Quarternary ingredients p.p.m.

ammonium cation Quaternary Ammonium 1500 1060 440 0 1060 0 1500 sequestrate Composition of this 100 71 29 53 124 44 153 invention Chlorhexidine digluconate 0 0 0 200 200 100 350 ion and 6.82 percent of the monobasic HEDTA anion.

To prepare the mono(chlorhexidine) di(I-IEDTA) salt it is necessary to add chlorhexidine in the molecular weight ratio of 505 parts chlorhexidine to 2 X 278 parts HEDTA measured in terms of free acid or 2 X 277 measured in terms of monobasic anion.

Thus 0.124 gm of chlorhexidine was dissolved in 2 ml These results show the superiority of the composition 55 of this invention to both the quaternary ammonium sequestrate and chlorhexidine digluconate as a bactericide.

EXAMPLE l7: Mono(chlorhexidine) di( I-IEDTA )/Myris tyldimethylbenzylammonium hydroxide/HEDTA salt a. Preparation of the composition By the same method as in Example 16, mono(chlorhexidine di(hydroxyethylethylenediaminetriacetate), containing 1 molar proportion of chlorhexidine to 2 molar proportions of HEDTA anion, was prepared by taking 2 m1 of the 15 percent solution of di(myristyldimethylbenzylammonium) mono( hydroxymethylethylenediaminetriacetate) and adding 0.08 gm of chlorhexidine. The product was a clear solution which could be further diluted with water.

b. Bactericidal tests The composition prepared in a) was tested for bactericidal activity in the same manner as in Example 1 1. The results are shown in Table X below.

TABLE X EXAMPLE l9: Mono(chlorhexidine) di(HEDTA)lDidecyldimethylammonium chloride Killing Dilution ppm ppm ppm ppm ppm of Total Quaternary Quaternary HEDTA Chlorhexidine active organic Ammonium Ammonium anion ingredients matter compound cation Quaternary ammonium cation Composition of a)above 400 283 1 17 108 391 508 By comparing these results with those for the quaternary ammonium sequestrate and chlorhexidine digluconate given in Table IX above, It Wlll be seen that the composition of the present invention is markedly superior to both these conventional bactericides.

EXAMPLE l8: Mono(chlorhexidine )mono-and di( HEDTA )/di(myristyldimethylbenzylammonium) mono(HEDTA) a. Preparation of the composition Using exactly the same method as in Example 16, compositions were prepared from the 15 percent w/w solution of di(myristyldimethylbenzylammonium) mono(hydroxyethylethylenediaminetriacetate) and chlorhexidine base which corresponded to mixtures of the mono(chlorhexidine) mono(HEDTA) salt and the mono(chlorhexidine) di(HEDTA) salt with excess of the di(quaternary ammonium) mono(HEDTA) salt.

Whereas in Example 16, the mono(chlorhexidine) mono (HEDTA) salt was made from 15 percent quaternary ammonium HEDTA salt and 8 percent chlorhexidine, and in Example 17 the mono (chlorhexidine) di( HEDTA) salt was made from 15 percent quaternary ammonium salt 4 percent chlorhexidine, in this Example the following mixtures were prepared:

a. 15% Quaternary HEDTA 1% chlorhexidine (i.e. less than 1:2) b. 15% Quaternary HEDTA 2% chlorhexidine (i.e. less than 1:2) 0. 15% Quaternary HEDTA 6% chlorhexidine (i.e. more than 1:2

but less than 1:1)

b. Bactericidal tests The compositions prepared in a) above were tested for bactericidal activity in the same manner as in Example 1 l. The results are shown in Table Xl below.

HEDTA in 1000 ml of distilled water.

Didecyldimethylammonium chloride is commercially available in the form of a concentrated solution containing 50 percent of the salt and 50 percent of water. A 0.2 percent solution of the salt was made up by dissolving 4.0 gm of this concentrate in 1000 ml of distilled water.

These two solutions were then mixed in the following proportions by volume:

Solution Quaternary ammonium Chlorhexidine salt di( HEDTA) A. (control) 100% 0 TABLE XI ppm ppm ppm ppm ppm of Chlorhexidine Total Quaternary Quaternary HEDTA Chlorhexidine active by weight of organic Ammonium Ammonium anion ingredients active matter compound cation Quarternary ingredients ammonium cation Solution a 1000 707 293 67 774 8.6 1067 b 500 354 146 67 421 16 567 c 150 106 44 60 166 36 210 By comparing these results with those for the quaternary ammonium sequestrate and chlorhexidine digluconate given m Table 1X above, it will be seen that the compositions of the present invention are markedly superior to both these conventional bactericides.

dine digluconate was also tested as a control. The re- 7 The results Obtained are shown in Table XIII below, sults are shown in Table XII below, and are also plotted and are also presented graphically in FIG. 2 of the acgraphically in FIG. 1 of the accompanying drawings. mpanying gs- TABLE XII Killing Dilutions Solution p.p.mtotal p.p.m. p.p.m. p. .m. p.p.m. p.p.m.

QAC and chloro- Chlorhex- HEDTA ammonium chloride gluconate hexadine di idine anion cation anion (HEDTA) A (control) 750 0 675 75 0 B 500 24 26 405 45 0 C 275 39 44 173 0 D 275 '64 71 I24 14 0 E 175 58 65 47 0 F 200 86 94 l8 2 0 200 95 I05 0 O 0 chlorhexadine digluconate (control) 400 228 0 0 0 172 These results show not only the superiority of mono (chlorhexidine) di(HEDTA) to chlorhexidine diglutonate as a bactericide but also the synergism which occurs on mixing mono (chlorhexidine) di(HEDTA) and didecyldimethylammonium chloride. If the bactericidal activities of the two compounds were simply additive, the test results would be along the line A, but in fact all the observations lie on a curve B lying wholly below A. Thus the bactericidal properties of the mixtures are greater than would be expected from the properties of the single components, and thus the mixtures are synergistic.

TABLE XIII Killing Dilutions Solution Total Concentration of Chlorhexidine HEDTA Quaternary Chloride bactericide ppm cation ppm anion ammonium anion ppm cation ppm A 300 I43 I57 0 0 B 400 I43 I57 92 8 C 500 I l9 13] 230 D l000 119 131 690 60 E (control) l000 0 0 920 80 It will be seen from these results that not only is mono(chlorhexidine) di(HEDTA) a much better bactericide than Hyamine but also that mixtures of the chlorhexidine sequestrate and the quaternary ammonium compound exhibit synergism-as in Example I). the curve B of the killing dilutions of mixtures of the two compounds always lies below the straight line A which shows the results which would be obtained if the bactericidal effects of the two compounds behaved additively (the tests indicated that the killing dilution of the quaternary ammonium compound alone was about 2500 ppm and the line A is drawn on this basis).

EXAMPLE 20: Mono(chlorhexidine) I EXAMPLE 2i: Mono(chlorhexidine) di(I-IEDTA)/Hyamine 10X 7 di(I-IEDTA)/l-Iyamine 1622 a. Preparation of the composition a. Preparation of the composition A 0.2 percent w/w solution of chlorhexidinedi(hydr- The 0.2 percent w/w solution of chlorhexidine di(- oxyethylethylenediaminetriacetate) was prepared by HEDTA) prepared in Example 20 was mixed with a 0.2 dissolving 0.954 gm of chlorhexidine base and 1.046 percent w/w solution of Hyamine 1622 (a United States gm of HEDTA in 1000 ml of distilled water and a 0.2 trade name for diisobutylphenoxyethoxyethyldimethylp r ent Solution of Hyamine 10X (a United States benzylammonium chloride) in the following ratios: trade name for di-isobutylcresoxyethoxyethyldimethylbenzylammonium chloride) in distilled water was also d up Solution Mono(chlorhexidine) Hyamine I622 di(HEDTA) The two solutions were then mixed in the following 53 ratios: A 100% 0% B 75% 25% Solution M iiiggfillillzolglilefpine) Hyamine 10X 5 E (control) 0% 10071 A 100% 071 B 7571 25% C 50% 50% D V 25 7, 75% In each case, clear, stable solutions resulted. E (control) '071 b. Bactericidal tests The compositions prepared in a) were tested for bacln a h Ca r. Stablfii Solutions r a tericidal activity in the same manner as in Example I l. b. Bactericidal Tests The results obtained are shown in Table XIV below,

The compositions prepa e n were tested for and are also presented graphically in FIG. 3 of the actericidal activity in the same manner as in Example I l. companying drawings.

TABLE XIV Solution Total concentration of Chlorhexidine HEDTA Quaternary I Chloride bactericide ppm cation ppm anion ammonium anion ppm cation 'ppm A 250 l 19 131 0 B 500 177 198 US '10 C 600 143 I57 276 24 D l0O0 ll9 l31 690 60 E (control) l000 0 0 920 80 It will be seen from these results that not only is mono(chlo I622. but also that mixtures of the chlorhexidine sequestrate in Example l9. the curve B of the killing dilutions of mixtures shows the results which would be obtained if the bacteric indicated that the killing dilution of the quaternary a on this basis).

EXAMPLE 22: Mono(chlorhexidine) di(HEDTA)/Cetylpyridinium chloride a. Preparation of the compositions The 0.2 percent w/w solution of chlorhexidine di( HEDTA) prepared in Example was mixed with 0.2 percent w/w solution of cetylpyridinium chloride in the following ratios. I I

In each case, clear, stable solutions resulted.

mmonium compound alone was about 2500 ppm and th rhexidine) di(HEDTA) a much better bactericide than Hyamine and the quaternary ammonium compound exhi of the two compounds always lies below the stra idal effects of the two compounds behaved additively (the tests bit synergism-as ight line A which e line A is drawn EXAMPLE 23: Chlorhexidine HEDTA sequestrates/coco-dihydroxyethyl-benzyl ammonium HEDTA mixtures a. Preparation of the compositions 1.68 Gm of a percent solution. of cocodi( hydroxyethyl')-benzylammonium chloride and 0.277 gm of hydroxyethylethylenediaminetriacetic acid were made up into 100 ml of solution (solution I) with distilled water. Thus the molar ratio of ammonium cation to HEDTA anion in the solution was 2:1. A similar solution (solution II) was prepared which contained, in addition 0.42 gm of chlorhexidine. These two solutions were then mixed in the following proportions:

Resultant solution Solu- Parts by Parts by by weight by weight by weight tion weight weight chlorhexidine ammonium HEDTA anion solutionsolution cation A l 0 0 0.769 0.277 B 5 l 0.07 4 0.769 0.277 C 2 l 0.14 0.769 0.277 D l l 0.2l 0.769 0.277 E l 2 0.28 0.769 0.277 F 0 l 0.42 0.769 0.277

b. Bactericidal tests 45 b. Bactericidal tests The compositions prepared ina) were tested for bactericidal activity in the same manner as in Example 1 l. .The results obtained are shown in Table XV below, and are also presented graphically in FIG. 4 of the accompanying drawings.

The compositions prepared in a) above were tested, .for bactericidal activity in the same way as in Example 1 1. To provide a control, chlorhexidine digluconate was also tested. The results are shown in Table XVI below.

mixtures of the two compou ample 19. the curve B of the killing dilutions of e two compoun results which would be obtained if the bactericidal effects ofth dilution of cetylpridinium' chloride alone was about 500 ppm a TABLE XV Solution Total Concentration of Chlorhexidine HEDTA anion Quaternary Chloride bactericide ppm cation ppm ppm ammonium amon cation ppm A 300 I43 157 0 0 B 400 143 157 10 C 600 143 I57 270 30 D 600 72 78 405 45 E (control) l000 0 0 900 It will be seen from these results that not only is monolchlorhexidine) di(HEDTA) a much better bactericide than cetylpyridimium chloride. but also that mixtures of the chlorhexidine sequestrate and the quaternary ammonium compound exhibit synergism-as in Exnd the line A is drawn on this basis).

TABLE XVI Killing Dilutions Solu- Total bacppm ppm ppm ppm tions tericide of quater- HEDTA chlorhexgluconate ppm nary ammoanion idine cation mum cation A 750 0 B 704 44 0 C 62 0 D 400 67 0 E 380 80 0 F 350 198 52 100 O Chlorhexidine digluconate 660 0 0 376 284 J These results show the supenority of compositions These results show the superiority of compositions containing the quaternary ammonium Cations, HEDTA containing the quaternary ammonium cations, HEDTA anions and chlorhexldine cations to both chlorhexidine anions and chlorhexidine cations to both chlorhexidine digluconate and quaternary ammonium sequestrates aS digluconate and quaternary ammonium sequestrates as bactericides. bactericides.

EXAMPLE 24: Chlorhexidine HEDTA EXAMPLE 25: Chlorhexidine HEDTA sequestrate/Dioctyldimethylammonium chloride sequestrate/Cetyltrimethyl-ammonium bromide mixtures mixtures a. Preparation of the composition 25 As shown above mono(chlorhexidine) mono Using the same technique as 1n Example 23, the fol- (HEDTA) is soluble in water at 20 C to the extent of lowing solutions were prepared: only 0.2 percent, and the mono(chlorohexhdine) di Solution l (HEDTA) is soluble to the extent of only about 0.3 per- Dioctyldimethylammonium chloride 0.65 gm cent. This is a serious disadvantage from the commergEP L- water to 8- in 30 cial point of view, and this Example shows that the solubility of these chlorhexldme sequestrates can be s luuonll greatly increased by adding cetyltrimethylammonium gg g yk y chkmde 8:2; g bromide, thus producing a commerciallly superior chlorhexidine gm Product- Distilled water to 100 Mixtures of chlorohexidine and HEDTA were added These two solutions were then mixed in the following and cetyltrimethylammonium bromide proportions: mide) was added until a clear solution was obtained.

Resultant Solution Solu- Parts by Parts by by weight by weight by weight tion weight weight chlorhexiammonium HEDTA anion solution solution dine cation A l 0 0 0.569 0.28 B 7 l 0.063 0.569 0.28 C 3 1 0.125 0.569 0.28 D l l 0.25 0.569 0.28 E l 2 0.33 0.569 0.28 F 0 l 0.5 0.569 0.28

b. Bactericidal tests Alternatively, the solutions can be prepared by adding The compositions produced in a) above were tested the solid chlorohexidine sequestrate to water and addfor bactericidal activity in the same manner as in Examing the ammonium compound until dissolution takes ple l l To provide a control, chlorhexidine digluconate place. was also tested. The results are shown in Table XVII In this way, the following solutions were prepared: below.

Solution Chlor- HEDTA Molar Cetyltri- TABLE XVIII hexadine anion ratio methyl cation by chlorammonium Killing dilutions by weight weight hexadine bromide HEDTA by weight Solution total ppm of ppm ppm ppm ppm quaternary HEDTA chlorgluconate A 14 0 20.0 bacterammonium anion hexidine anion B 116 icide canon canon excess C 7.2 7.l 122 20.7 A 50 500 250 0 0 D 5.5 8.3 1:2 ll.l B 483 300 I50 33 0 excess C 516 300 l50 66 0 D 421 217 108 96 0 E 423 100 123 0 All these solutions were clear and stable. 398 167 83 148 0 b. Bactericidal tests g The solutions produced in a) were tested for bacteridigluccidal activity by the Anti-septic Test described in Exonate 660 0 0 376 284 ample 11 above. These tests employed Pseudomonas pyocyanea in:-

A. Standard hard water (W.H.O.formulation de- S l I, Ch] DTPA C t ,d

scribed in Example 12 above), without the addition u g z by 25; g ut i of serum; cation B. Distilled water with the addition of 10% bovine by weght Serum; and A 10.5 7.5 15.0

C. Standard hard water (W.H.O formulation), with B 195 the addition of 10% of bovine serum.

To provide a control, chlorhexidine digluconate was The resulting solutions were clear. also tested. The results are shown 1n Table XVIII below b. Bactericidal tests.

' The solutions prepared in a) were tested for bacteri- TABLE XVIII cidal activity in the same manner as in Example 25, in:-

A. Standard hard water (W.H.O. formulation), with Killing dilutions no serum:

B. Distilled water, with 10 percent of bovine serum; A) Standard hard water, no serum and Solution ppm chlorppm HEDTA ppm ppm Cetri- C. Standard hard water (W.H.O formulation) With 10 'f percent of bovine serum.

cation conate To provlde a control, chlorhexidine digluconate was A 80 45 0 114 also tested. The results are shown in Table XIX below. B 200 100 O 289 C 100 100 O 288 D 80 120 0 162 TABLE XIX Chlorhexidine digluconate 570 O 430 0 Killing dilutions B) Distilled water, 10% serum A) Standard hard water, no serum Solution ppm chlorppm HEDTA ppm ppm Cetri- Solution ppm chlorppm DTPA ppm ppm Cetrihexidine anion glumide hexidine anion glumide cation conate cation conate A 130 70 0 185 A 60 0 0 86 B 100 50' 0 145 B 67 33 0 98 c 50 50 0 144 hi q D 60 9O 0 121 me Chlob digluconate 570 0 430 O hexidine 5 digluconate 2 5 0 2 5 B) Distilled water, 10% serum C) Standard hard water, 10% serum Solution ppm l ppm PTPA ppm ppm hexidine amon glumide Solution ppm chlorppm HEDTA ppm ppm Cetricanon conate hexadine anion glumide 40 A 90 60 O 129 cation conate B O A 320 180 0 456 B 760 390 0 1097 C 390 380 0 H24 digluconate 285 0 215 0 a 200 300 0 405 5 C) Standard hard water, 10% serum hexidine Solution ppm chlorppm DTPA C ppm ppm etri digluconate 1,145 0 855 0 hexidine anion mide cation conate A 300 200 0 430 B 670 330 O 9 0 These results show that, by lncorporatmg cetyl- Chlop 8 trimethyl-ammonium bromide and a sequestering hexidine digluconate l 145 0 855 0 aminocarboxylic acid into compositions containing chlorohexidine, the amount of chlorhexidine needed to produce a given bactericidal effect can be greatly reduced. In view of the fact that HEDTA and cetrimide are both far, far cheaper than chlorohexidine, this enables an enormous reduction in the cost of the bactericidal composition needed to produce a given effect. A1- ternatively, the efficacy of the bactericidal composition which can be produced at any given cost may be greatly increased. 4

EXAMPLE 26: Chlorhexidine DTPA Sequestrate/Cetrimide mixtures.

a. Preparation of the compositions Using the same method as in Example 25, the following chlorhexidine DTPA sequestrate/cetrimide mixtures were prepared:-

EXAMPLE 27: Efficacy of compositions of this invention against various bacteria In order to show that the efficacy of the compositions of this invention is not restricted to Pseudomonas pyocyanea, solution A from Example 25 and solution A from Example 26 were tested with chlorhexidine digluconate, for their killing power against a wide range of bacteria. The tests were conducted in the same manner These results Show that h Compositions of this as in Example 11 in distilled water containing 10 vention are markedly superior to chlorhexidine diglucent of bovine serum, and the bacteria used were as folconate against a wide range of bacterla' lows:- EX

AMPLE 28: 1. Pseudomonas pyocyanea N.C.T.C. 6749 To confirm the results with E.Coli shown in Example 2' Proteus vulgaris 4635 27, tests of the bactericidal properties of compositions of this lnvention against E.coli were carried out by the 3 Salmonella hi N C T C 3390 method of British Standard 3286:1960 described above w in standard hard water (W.H.O formulation) contain- 4. Escherichia coli N.C.T.C. 8196 mg percent of bovine serum. The solutions used were as follows, chlorhexidine digluconate being pro- 5. Staphylococcus aureus N.C.T.C. 3750 vided as a control:-

Solution by weight by weight by weight by weight by weight weight chlorhexidine HEDTA anion DTPA anion NTA anion gluconate Cetrimide cation anion A 13.0 7.2 O O O 18.9 B 13.6 0 6 5 O 0 l 1.3 C 14.4 0 0 5.1 O 15.4 Chlorhexidine O 8.6 O

digluconate v (control) 1 1.4 0 0 0 8.6 O The compositions were tested at concentrations equivalent to 100, 150, 200, 250, 300, 400, 500, 600, and 800 p.p.m. of chlorhexidine sequestrate or digluconate. The results, which are shown in Table XXI below, are expressed in terms of the number of survivors per million bacteria inoculated.

TABLE XXI Solution Dilution chlorhexidine in ppm A B C digluconate chlorhexi- (control) dine sequestrate Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 100 /c 150 137 24 85 432 36 u/c 200 190 78 27 260 O 30 0 O 250 0 O 0 300 0 0 0 113 0 0 0 44 0 u/c 400 0 0 0 0 O 0 O O 0 133 162 500 0 O 0 0 0 O 0 O 0 174 1 10 600 0 0 0 O 0 O 0 0 0 100 0 23 800 0 0 0 0 0 0 0 0 0 O 0 0 u/c survivors too numerous to be counted These results confirm that the compositions of this invention are markedly superior as bactericides against E.coli as compared with chlorhexidine digluconate.

The results obtained are shown in Table XX below. w l i 1. A salt selected from the group consisting of: TABLE XX mono-chlorhexidine nitrilotriacetate,

Killing dilution tri( chlorhexidine) di(diethylenetriaminepentaace- Solution ppm chlorhexidine sequestrate tate) )py )pgyp P mono-chlorhexidine N,N-dihydroxyethylaminoace- C011 tate,

Example 25 sohmon A 200 300 100 150 200 mono chlorhexidine N hydroxyethyle thylenediamlnetriacetate, and Example 26 Solution A 150 350 100 200 100 mono-chlorhexidine d1[N-hydroxyethylechlorhexidine thylenediammetriacetate digluconate 500 750 300 300 500 

1. A SALT SELECTED FROM THE GROUP CONSISTING OF: MONO-CHLORHEXIDINE NITRILOTRIACETATE, TRI(CHLORHEXIDINE) DI(DIETHYLENETRIAMINEPENTAACETATE) MONO-CHLORHEXIDINE N,N-DIHYDROXYETHYLAMINOACETATE, MONO-CHLORHEXIDINE HYDROXYETHYLETHYLENEDIAMINETRIACETATE, AND MONO-CHLORHEXIDINE DI(NHYDROXYETHYLETHYLENEDIAMINETRIACETATE). 